Preparation of Polycaprolactone Nanoparticles Via Supercritical Carbon Dioxide Extraction of Emulsions

Drug Deliv. and Transl. Res. DOI 10.1007/s13346-017-0422-3

ORIGINAL ARTICLE

Preparation of polycaprolactone nanoparticles via supercritical carbon dioxide extraction of emulsions

Adejumoke Lara Ajiboye1 & Vivek Trivedi1 & John C. Mitchell1

# The Author(s) 2017. This article is an open access publication

Abstract Polycaprolactone (PCL) nanoparticles were pro- rapidly increased over the years because they can successfully duced via supercritical fluid extraction of emulsions (SFEE) maximise the efficacy of the drug molecules [1]. using supercritical carbon dioxide (scCO2).Theefficiencyof Nanoparticles are particles with a diameter range from 1 to the scCO2 extraction was investigated and compared to that of 100 nm, even though the dynamic range can cover the whole solvent extraction at atmospheric pressure. The effects of pro- nanometre scale [2]. As a drug delivery system, nanoparticles cess parameters including polymer concentration (0.6–10% can entrap drugs or biomolecules into their internal structures w/w in acetone), surfactant concentration (0.07 and 0.14% and/or adsorb these drugs or biomolecules onto their external w/w) and polymer-to-surfactant weight ratio (1:1–16:1 w/w) surfaces [3]. on the particle size and surface morphology were also inves- Due to their particle size, nanoparticles can move freely tigated. Spherical PCL nanoparticles with mean particle sizes through the body via the smallest capillary vessels, cell and between 190 and 350 nm were obtained depending on the tissue gaps in order to reach their target organs [4]. These polymer concentration, which was the most important factor properties of nanoparticles help to modify the bio- where increase in the particle size was directly related to total distribution and pharmacokinetic properties of the adsorbed/ polymer content in the formulation. Nanoparticles produced entrapped drug molecules leading to an improvement in the were analysed using dynamic light scattering and scanning efficacy of the drug, decrease in unwanted side-effects and electron microscopy. The results indicated that SFEE can be increase in patient compliance [5]. Nanoparticles can be pre- applied for the preparation of PCL nanoparticles without ag- pared from inorganic or polymeric materials. Polymeric nano- glomeration and in a comparatively short duration of only 1 h. particles are more common and appropriate as they can be chemically modified to be biodegradable and biocompatible Keywords Solvent extraction . Polycaprolactone . [6]. Biodegradable substances are broken down in vivo either Nanoparticles . Supercritical carbon dioxide . Supercritical enzymatically or non-enzymatically or both, to give rise to fluid extraction of emulsions toxicologically safe by-products which are further removed by the normal metabolic pathways. The use of biodegradable polymers has greatly increased over the past decade, and they Introduction can generally be classified as either (1) synthetic biodegradable polymers which include relatively hydrophobic materials for example poly-lactic-co-glycolic acid (PLGA), The use of drug delivery systems, particularly the micro- and polycaprolactone (PCL) and others, or (2) naturally occurring nano-scale intelligent systems for therapeutic molecules has polymers such as chitosan, hyaluronan, etc. [7]. Principally, synthetic polymers have many inherent advantages since their * Vivek Trivedi structures can be manipulated to generate specialised carriers [email protected] to suit particular applications [8]. PCL is a semi-crystalline polyester that is hydrophobic, 1 Faculty of Engineering and Science, University of Greenwich, biodegradable and biocompatible. When compared to other Central Avenue, Chatham Maritime, Kent ME4 4TB, UK polymers, the biodegradation of PCL is slow; hence, it can Drug Deliv. and Transl. Res. be highly suitable for the design of controlled release delivery SFEE have an ordered size and morphology because emulsifi- systems [9–11]. The glass transition temperature (Tg)of cation process provides a template and the fast kinetics of the − 60 °C and low melting point (59–64 °C) of PCL allows scCO2 extraction prevents particle agglomeration [22]. for the easy fabrication of delivery systems at reasonably The aim of this work was to produce polymeric nanoparticles low temperatures [10]. Furthermore, PCL has an excellent by employing scCO2 extraction of the solvent in the organic blend compatibility with other polymers which facilitates tai- phase of nano-emulsions prepared via spontaneous emulsifica- loring of desired properties like degradation kinetics, hydro- tion. The major objective of this work was to study the suitability philicity and mucoadhesion [12, 13]. of SFEE and the effect of various parameters influencing the In the last decades, PCL polymers have been a key area of preparation of nanoparticles using PCL as a model polymer. interest in the development of controlled delivery systems espe- The effect of parameters including polymer and surfactant con- cially for peptides and proteins [14]. These systems using PCL centration on the particles’ hydrodynamic diameter, polydisper- or PCL blends have shown to be useful in varied conditions. For sity index (PDI) and zeta potential was investigated. The time example, while developing PCL microspheres for vaccine de- and the optimum CO2 flow rate were also determined for the livery, Jameela et al. showed that PCL has good permeability to complete extraction of oil phase/solvent at both high pressures in proteins and contrary to poly (lactic acid) PLA and poly glycolic scCO2 and atmospheric conditions. acid (PGA), PCL degrades very slowly and does not give rise to an acidic environment which can negatively affect the antigenic- ity of the vaccine, and thus, it can be used as a vaccine carrier Experimental section [15]. Observations by Youan et al. also suggested that PCL delivery systems are unaffected by simulated gastric fluid and Materials hence could give some protection to the encapsulated peptide from proteolytic destruction in the stomach, allowing the PCL (Mw = 45,000 Da) was obtained from Shenzhen ESUN, entrapped drugs to pass unharmed into the intestine for presen- China, and Tween 80 and acetone were purchased from Sigma tation to the gut-associated lymphoid system [16]. Aldrich, UK. Liquid CO2 at 99.9% was supplied by BOC Ltd., Various techniques can be employed to prepare nanoparticles, UK. All chemicals were of analytical grade and used without many of which involve a type of emulsion evaporation. The any further purification. Deionised water was used throughout preparation of nano-emulsions via spontaneous emulsification the study. mechanism using a water-miscible solvent is an established method that allows for the generation of nano-sized droplets Methods [17]. Nano-emulsions are made of fine oil-in-water dispersions with droplet sizes ranging from 100 to 600 nm. They can be Oil-in-water emulsion preparation prepared using a small surfactant concentration and they typical- ly exhibit low oil-water interfacial tensions. Spontaneous emul- The nano-emulsions were prepared through spontaneous emulsification occurring when the organic and aqueous phases are in sification mechanism. The method used for the preparation of contact determines the droplet size and size distribution, and PCL emulsions was adapted from the work reported by Prieto depends on variables including bulk viscosity, surfactant con- and Calvo [22]. The organic phase consists of a homogenous centration and structure [17–19]. In this work, the organic phase solution of PCL in acetone and Tween 80, while deionised water consists of a homogenous solution of the polymer in a water- made up the aqueous phase. In summary, an appropriate quantity miscible solvent (acetone) and surfactant which is mixed with of PCL was first dissolved in acetone (0.6–10% w/w)inather- the aqueous phase to produce nano-emulsion. mostatic water bath at 40 °C, before the addition of surfactant in Although emulsification of polymer mixtures is known to be a centrifuge tube to obtain required concentrations (0.07 and an efficient method, it can be associated with limitations 0.14%) or ratios (1:1–16:1 w/w). The contents in the tube were concerning process efficiency and control of the particle size mixed together using a vortex mixer for 1 min. After vortexing, and distribution when particles are being recovered from the 35.8 g of warm deionised water (40 °C) was added, and the tube emulsion through conventional processes [20]. Supercritical flu- was placed back on the vortex mixer for 5 min in order to form a id extraction of emulsion (SFEE) is a novel particle formation homogenous emulsion. Solvent extraction was carried out im- technique where it employs a supercritical fluid (SCF) such as mediately after emulsion was prepared. supercritical carbon dioxide (scCO2) to rapidly extract the solvent or oil phase of an emulsion. The removal of the solvent Solvent extraction via SFEE leads to the precipitation of the solute resulting into an aqueous suspension containing nanoparticles. Particles can thereafter be SFEE was conducted in a batch mode where 25-g emulsion was recovered from the aqueous suspension by centrifugation and/or introduced in the high-pressure vessel (Thar Process Inc., USA) by evaporation at room temperature [21]. Particles generated via pre-heated to 40 °C (± 2 °C), the vessel was then closed and Drug Deliv. and Transl. Res.

liquid CO2 was pumped until a pressure of 100 bar was Results and discussion achieved. The system was left to equilibrate under stirring for

15 min before continuously flushing with fresh CO2 for 1 h. The Solvent extraction initial experiments were carried out at controlled scCO2 flow rates of 1, 4 or 10 g/min to determine the extraction efficiency The rate of acetone extraction via supercritical processing was of scCO2. The system was then depressurised at a rate of 6 bar/ evaluated. The temperature (40 °C) and pressure (100 bar) min, and the sample was removed from the high-pressure vessel. selected for SFEE were based on the low melting point (59– The acetone extraction efficiency was calculated using the 64 °C) of the PCL polymer and the good miscibility of acetone weight difference before and after the procedure. with scCO2 at these conditions [23]. Figure 1 shows that at a constant exposure time of 1 h, the percentage weight of acetone extracted from the emulsion increases with an increasing Solvent extraction at atmospheric pressure flow rate of scCO2. Complete removal of acetone occurred after 1 h at a CO2 flow rate of 10 g/min. Extraction of the Solvent extraction at atmospheric pressure was conducted at organic phase can occur either by diffusion mechanism in the 40 °C (± 2 °C) in a thermostatic water bath (Fisher Scientific, aqueous phase or by direct extraction upon contact between UK) with 25-g emulsion in a 50-ml beaker, at a stirring rate of acetone and scCO2 in the emulsion droplet. Thus, acetone 300 rpm. The evaporation of acetone was determined at time 0, extraction at higher scCO2 flow rates improves as it allows 1, 2, 3, 4 and 24 h using weight difference similar to SFEE. for a comparatively large quantity of CO2 to be in contact with the emulsion and consequently the organic phase [24]. Physicochemical characterisation of nanoparticle The rate of acetone extraction at atmospheric conditions wasalsoevaluatedandcomparedtoscCO2 conditions. Particle size and zeta potential measurement Acetone extraction at atmospheric pressure was carried out at 40 °C to obtain comparative data at the same temperature These measurements were carried out in order to determine used in supercritical fluid extraction. Also, the conditions used the size and surface charge of the nanoparticles. The average in these experiments allowed the work to be carried at a tem- – particle sizes and polydispersity index (PDI) were measured perature below the melting point of PCL (59 64 °C) to avoid by dynamic light scattering (DLS) using a Zetasizer Nano-ZS possible particle deformation due to simultaneous melting of (Malvern Instruments Ltd., UK). The instrument was the polymer. The complete removal of acetone at atmospheric equipped with a laser emitting at 633 nm, and backscattering pressure (Fig. 2) could only be obtained after 24 h of contin- detection was set at an angle of 173°. Samples were diluted in uous stirring at 40 °C. deionised water (1 in 4) and measurements were carried out in Moreover, the extraction at atmospheric pressure also led to triplicate at room temperature (25 °C). The mean particle size the agglomeration of particles (Fig. 2, inset) consequently de- was the average of three independent measurements. The zeta creasing the total product yield. The gas-like viscosity of scCO2 potential (Z-potential) of the aqueous dispersions was also coupled with high diffusivity facilitates the rapid and efficient determined using Zetasizer Nano-ZS at 25 °C. The measure- extraction of acetone in comparison to solvent extraction at ments were carried out in triplicate on the same samples used atmospheric pressure at the same temperature without particle for particle size measurements. agglomeration [25]. Based on these findings (rapid extraction timeandnoaggregationatscCO2 conditions), SFEE was cho- sen as the method for solvent removal for the rest of the study.

Particle morphology The extraction conditions were kept constant with a scCO2 flow rate of 10 g/min for 1 h at 100 bar and 40 °C. Scanning electron microscopy (SEM) was performed in order to determine the shape and surface morphology of the nanoparticles. The frozen aqueous nanoparticle dispersions were freeze- dried under vacuum at − 55 °C using a ScanVac CoolSafe freeze dryer (LaboGene ApS, Denmark). Approximately 1 mg of the dried particles was placed on the sample stub using a carbon adhesive and the extra loose particles were removed. The sample was then coated with chromium, thereafter the stub was mounted onto the sample holder and placed inside the Hitachi SU8030 (Hitachi High-Technologies, UK) scanning electron microscope. Micrographs were collected using the upper detec- tor at a voltage of 1.0 kV. Fig. 1 Acetone extraction via SFEE Drug Deliv. and Transl. Res.

Table 1 PDI and Z-potential of PCL particles before and after extraction of a 25-g emulsion (0.6–10% w/w PCL, 28.3% w/w of acetone, 71.6% w/w of distilled water and 0.07% w/w surfactant)

PCL concentration PDI ± SD PDI ± SD Z-potential (mV) ± (% w/w in acetone) before SFEE after SFEE SD after SFEE

0.6 0.13 ± 0.07 0.11 ± 0.10 −10 ± 1 1 0.19 ± 0.06 0.09 ± 0.07 −12 ± 1 2 0.09 ± 0.07 0.16 ± 0.08 −15 ± 2 4 0.17 ± 0.04 0.22 ± 0.04 −24 ± 1 6 0.23 ± 0.07 0.27 ± 0.04 −21 ± 0 8 0.21 ± 0.05 0.27 ± 0.02 −24 ± 1 10 0.21 ± 0.04 0.25 ± 0.02 −26 ± 1 Fig. 2 Acetone extraction at atmospheric pressure. Inset:animageof typical agglomerate seen after acetone extraction at 40 °C

Effect of polymer concentration nanoparticles [27, 28]. The results also show a marked difference between the size before and after acetone extraction, PCL content was varied between 0.6 and 10% w/w, and the which could be due to the presence of voids in the polymer effects of polymer concentration on the morphological char- matrix which collapse upon the extraction of acetone by acteristics and surface charge of the particles were scCO2. It is well known that free spaces within the polymer investigated. network caused by the presence of a solvent can cause parti- As noted in Fig. 3, at a constant surfactant concentration cles to shrink and reduce in size after drying [29]. (0.07% w/w) and water-to-acetone ratio (2.5:1), an increase in The particle surface charge was determined by carrying out polymer concentration results in an increase in particle size. Z-potential measurements. Table 1 presents the Z-potential These results are in line with findings of Santos et al. [26]and values of PCL nanoparticles prepared with different polymer Kwon et al. [27]. Primarily, it is assumed that nanoparticle concentrations. Nanoparticles produced have a negative sur- formation occurs when both the organic and aqueous phases face charge which can be as a result of the carbonyl group of are in contact. The solvent diffuses from the organic part into the PCL polymer present at the surface of the nanoparticle the aqueous and takes along with it some polymer chains structure. Table 1 also shows a shift in Z-potential values with which are still in solution, then as the solvent spreads further an increasing polymer concentration. The change in Z-poten- into the water, the accompanying polymer chains aggregate tial can be attributed to the shielding effect by the surfactant forming nanoparticles [28]. A rise in the polymer content in molecules at the interface due to interaction with PCL. the organic phase consequently increases both its hydrophobic Therefore, as the polymer content increases at a constant sur- composition and viscosity which is relative to a higher mass factant concentration, the number of Tween 80 molecules transfer resistance, hence leading to a negative effect on the available at the interface decreases consequently resulting in distribution efficiency of the polymer-solvent composition in- a reduced shielding effect and leading to a change in Z-poten- to the external aqueous phase and the formation of large tial [30]. Table 1 also presents PDI of produced nanoparticles

700 700 600

600 )mn(ezistelpord 500 500 400 400 300 0.07% conc. 300 Droplet size 0.14% conc. n Parcle size aeM 200

Average size (nm) 200 100 100 0 0 0.6 1 2 4 6 8 10 0.6 1 2 4 6 8 10 PCL concentraon (%w/w in acetone) PCL concentraon (% w/w in acetone) Fig. 4 Comparison of the mean emulsion droplet size (nm) of 0.07 and Fig. 3 The effect of PCL concentration in the organic phase on the 0.14% w/w surfactant concentrations at a constant PCL concentration average sizes before and after SFEE (% w/w in acetone) Drug Deliv. and Transl. Res.

450 1200 400 1000 350

300 Average droplet size (nm) 800 250 1:1 rao 200 0.07% conc. 600 2:1 rao 150 0.14% conc. 4:1 rao 400 100 8:1 rao Mean parcle size (nm) 50 200 16:1 rao 0 0 0.6 1 2 4 6 8 10 0 2 4 6 8 10 12 PCL concentraon (% w/w in acetone) PCL concentraon (% w/w in acetone) Fig. 5 Contrast of the mean particle size (nm) of 0.07 and 0.14% w/w Fig. 6 Mean emulsion droplet size at a varying polymer-to-Tween 80 surfactant concentrations after SFEE at a constant PCL concentration (% ratio (1:1–16:1) and a constant PCL concentration w/w in acetone) at various polymer concentrations. In general, the PDI was to the role of Tween 80 in the emulsion. A low concentration very low for all formulations, confirming that the prepared of surfactant in the emulsion and consequently at the solvent- nanoparticles are highly monodisperse and have narrow par- water interfacial layer would mean that there is not enough ticle size distribution. A slight increase in PDI was observed surfactant to cover the surface of drops, resulting in coales- with the increase in polymer concentration and particle size. cence of droplets and increase in size. An increase in the This could be attributed to the increase in viscosity of the surfactant concentration would therefore result in a higher organic phase and possible coalescence of smaller droplets number of surfactant molecules present at the interfacial layer into larger droplets at higher concentrations resulting in com- promoting the formation of smaller droplets [34]. paratively broad particle size distribution [31, 32]. Similar to earlier observations, an increase in polymer concentration at a constant surfactant concentration generally re- Effect of surfactant concentration sulted in an increase in particle size. As noted when Figs. 4 and 5 are compared, there is also a reduction in size after In order to investigate the effect of surfactant on the particle acetone extraction by SFEE. size, PDI and surface charge, at a constant polymer concen- Figure 5 also shows that a higher surfactant concentration tration, the surfactant content was doubled from 0.07 to 0.14% led to decrease in particle size and standard error. This result is w/w. similar to that reported by Mu and Feng [35]. The reduction in As observed in Fig. 4, there is a general increase in droplet size can also be anticipated from the role of surfactant as an size as polymer concentration increases. Furthermore, an in- emulsion stabiliser in the formation of nanoparticles [36]. crease in surfactant concentration resulted in smaller droplet Z-potential measurements (Table 2) were determined on size which was more evident at higher polymer concentra- 0.14% w/w Tween 80 samples to understand the effect of the tions. This result is similar to the findings from the work done surfactant concentration on the particle surface charge. Similar by Goloub and Pugh [33]. The positive effect of a higher to earlier results, Z-potential increased with the polymer con- Tween 80 concentration on droplet size can be easily related centration but no significant change could be observed with the higher surfactant content in these samples.

Table 2 PDI and Z-potential of PCL nanoparticles before and after 700 – SFEE of a 25-g emulsion (0.6 10% w/w,28.3%w/w of acetone, 71.6% 600 w/w of distilled water and 0.14% w/w surfactant) )m n( n( 500 ezis e ezis PCL concentration PDI ± SD PDI ± SD Z-potential (mV) 1:1 rao

lcitrap 400 (% w/w in acetone) before SFEE after SFEE ± SD after SFEE 2:1 rao 300 4:1 rao 0.6 0.09 ± 0.03 0.11 ± 0.05 −14 ± 1 egare 8:1 rao 1 0.14 ± 0.05 0.19 ± 0.04 −17 ± 3 200

v 16:1 rao 2 0.09 ± 0.04 0.14 ± 0.07 −19 ± 3 A 100 4 0.14 ± 0.08 0.19 ± 0.08 −22 ± 2 0 6 0.16 ± 0.06 0.22 ± 0.12 −25 ± 3 024681012 8 0.19 ± 0.04 0.26 ± 0.09 −29 ± 3 PCL concentraon (% w/w in acetone) 10 0.19 ± 0.06 0.24 ± 0.05 −29 ± 3 Fig. 7 Mean particle size after SFEE at a varying polymer-to-Tween 80 ratio (1:1–16:1) and a constant PCL concentration Drug Deliv. and Transl. Res.

a b

Fig. 8 Example of SEM micrographs of particles processed by SFEE. a 0.6% w/w. b 10% w/w at ×20,000 magnification

Like the previous results, the PDI recorded for the higher shape and surface morphology of produced particles. surfactant concentration samples (Table 2) are very low for all Figure 8 presents an example of micrographs of PCL nano- the preparations and they indicate that the nanoparticles pro- particles prepared by SFEE. duced have a uniform particle size distribution. From the SEM micrographs, it is clearly seen that the particles prepared by SFEE are spherical and generally with uni- Effect of polymer-to-surfactant weight ratio form particle size distribution. SEM micrographs also confirm DLS results that there is an increase in particle size at a higher To further study the effect of varying surfactant/polymer con- polymer concentration. tent on nanoparticle formation, the concentrations were altered to prepare emulsions with polymer-to-surfactant weight ratios of 1:1, 2:1, 4:1, 8:1 and 16:1. Conclusion As shown in Figs. 6 and 7, similar to previously discussed findings, at a constant polymer-to-surfactant weight ratio, in- Spherically shaped monodisperse polycaprolactone nanoparti- creasing polymer concentration led to the formation of larger cles have been successfully prepared by supercritical fluid extrac- particles. Interestingly, 1:1 w/w ratio samples have noticeably tion of emulsions without agglomeration and in a comparatively higher particle size in comparison to the rest of the data sets. short duration. Varying the polymer concentration between 0.6 This could be attributed to the depletion effect from non- and 10% w/w during the formulation stage allowed for a control adsorbing Tween 80 micelles at such high surfactant concen- of the particle size of the resulting polycaprolactone nanoparti- tration [37]. The presence of free micelles at high surfactant cles. An increase in the emulsion surfactant concentration from concentrations can cause increase in osmotic pressure in the 0.07 to 0.14% w/w at a constant polymer concentration led to a liquid surrounding the particles to an extent which can result decrease in particle size. There was minimal change in size when in particle aggregation [38]. the polymer-to-surfactant weight ratio was varied between 2:1 It is also important to note that the error associated to the and 16:1 w/w ratios. 1:1 w/w polymer-surfactant weight ratio particles prepared with 1:1 w/w ratio is also very high in com- produced polycaprolactone nanoparticles with sizes between parison to other formulations which suggest flocculation/ 200 and 500 nm with high standard error between repeats. aggregation of prepared particles. There were minimal changes Although preliminary studies have shown that these in particle sizes at polymer-to-surfactant weight ratio from 16:1 polycaprolactone nanoparticles can be efficiently produced to 2:1 w/w. These results suggest that it may be possible to pre- via supercritical fluid extraction of emulsions, further studies pare PCL nanoparticles in wide ranging sizes using extremely investigating their potential to be used as delivery vehicles high surfactant-to-polymer ratio but factors mentioned above are either by surface adsorption of a drug or encapsulation within important to be considered. However, particle sizes ranging from to provide modified release need to be carried out. 150 to 350 nm with narrow distribution can be reproducibly obtained using low to moderately high surfactant concentrations. Compliance with ethical standards

Particle morphology Conflict of interest The authors declare that they have no conflict of interest.

SEM micrographs were obtained using chromium-coated Open Access This article is distributed under the terms of the Creative freeze-dried PCL nanoparticles in order to investigate the Commons Attribution 4.0 International License (http:// Drug Deliv. and Transl. Res. creativecommons.org/licenses/by/4.0/), which permits unrestricted use, 19. Shahidzadeh N, Bonn D, Aguerre-Chariol O, Meunier J. distribution, and reproduction in any medium, provided you give appro- Spontaneous emulsification: relation to microemulsion phase be- priate credit to the original author(s) and the source, provide a link to the haviour. Colloids Surf A Physicochem Eng Asp. 1999;147:375. Creative Commons license, and indicate if changes were made. 20. Falk RF, Randolph TW. Process variable implications for residual solvent removal and polymer morphology in the formation of gentamycin-loaded poly(L-lactide) microparticles. Pharm Res. 1998;15:1233. References 21. Rao JP, Geckeler KE. Polymer nanoparticles: preparation techniques and size-control parameters. Prog Polym Sci. 2011;36:887. 1. Guilherme MR, Mauricio MR, Tenório-Netoa ET, Kunita MH, 22. Prieto C, Calvo L. Supercritical fluid extraction of emulsions for the Cardozo-Filho L, Cellet T, et al. Polycaprolactone nanoparticles production of vitamin E nanocapsulates. XXI Int Conf Bioencapsul. containing encapsulated progesterone prepared using a scCO2 2013;1:14. emulsion drying technique. Mater Lett. 2014;124:197. 23. Katayama T, Ohgaki K, Maekawa G, Goto M, Nagano T. 2. Psarros C, Lee R, Margaritis M, Antoniades C. Nanomedicine for Isothermal vapor-liquid equilibria of acetone-carbon dioxide and the prevention, treatment and imaging of atherosclerosis. Maturitas. methanol-carbon dioxide systems at high pressures. J Chem Eng 2012;73:52. Jpn. 1975;8:89. 24. Campardelli R, Baldino L, Reverchon E. Supercritical fluids appli- 3. Liu G, Liu W, Dong CM. UV- and NIR-responsive polymeric cations in nanomedicine. J Supercrit Fluids. 2015;101:193. nanomedicines for on-demand drug delivery. Polym Chem. 25. Campardelli R, Porta GD, Reverchon E. Solvent elimination from 2013;4:3431. polymer nanoparticle suspensions by continuous supercritical ex- 4. Liu Z, Jiao Y, Wang Y, Zhou C, Zhang Z. Polysaccharides-based traction. J Supercrit Fluids. 2012;70:100. nanoparticles as drug delivery systems. Adv Drug Deliv Rev. 26. Santos DT, Martín A, Meireles MA, Cocero MJ. Production 2008;60:1650. of stabilized sub-micrometric particles of carotenoids using 5. Gonçalves C, Pereira P, Gama M. Self-assembled hydrogel nano- supercritical fluid extraction of emulsions. J Supercrit Fluids. particles for drug delivery applications. Materials. 2010;3:1420. 2012;61:167. 6. Yang YY, Wang Y, Powell R, Chan P. Polymeric core-shell nanopar- 27. Kwon H, Lee J, Choi S, Jang Y, Kim J. Preparation of ticles for therapeutics. Clin Exp Pharmacol Physiol. 2006;33:557. PLGA nanoparticles containing estrogen by emulsification– 7. Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as bio- diffusion method. Colloids Surf A Physicochem Eng Asp. degradable controlled drug delivery carrier. Polymers. 2011;3:1377. 2001;182:123. 8. Crucho C, Barros MT. Formulation of functionalized PLGA poly- 28. Galindo-Rodriguez S, Allémann E, Fessi H, Doelker E. meric nanoparticles for targeted drug delivery. Polymer. 2015;68:41. Physicochemical parameters associated with nanoparticle forma- 9. Seremetaa KP, Chiappettaa DA, Sosnik A. Poly (ɛ-caprolactone), tion in the salting-out, emulsification-diffusion, and Eudragit® RS 100 and poly(ɛ-caprolactone)/Eudragit® RS100 nanoprecipitation methods. Pharm Res. 2004;21:1428. blend submicron particles for the sustained release of the antiretro- 29. Pasquali I, Bettini R. Are pharmaceuticals really going supercritical. viral efavirenz. Colloids Surf B: Biointerfaces. 2013;102:441. Int J Pharm. 2008;364:176. 10. Woodruff MA, Hutmacher DW. The return of a forgotten polymer- 30. Asasutjarit R, Sorrachaitawatwong C, Tipchuwong N, Pouthai S. polycaprolactone in the 21st century. Prog Polym Sci. 2010;35:1217. Effect of formulation compositions on particle size and zeta poten- 11. Karuppuswamy P, Venugopal JR, Navaneethan B, Laiva AL, tial of diclofenac sodium-loaded chitosan nanoparticles. Int J Med Ramakrishna S. Polycaprolactone nanofibers for the controlled re- Health Biomed Bioeng Pharm Eng. 2013;7:568. lease of tetracycline hydrochloride. Mater Lett. 2015;141:180. 31. Sharma N, Madan P, Lin S. Effect of process and formulation var- 12. Bilensoy E, Sarisozen C, Esendagli G, Dogan AL, Aktas Y, Sen M, iables on the preparation of parenteral paclitaxel-loaded biodegrad- et al. Intravesical cationic nanoparticles of chitosan and able polymeric nanoparticles: a co-surfactant study. Asian J Pharm polycaprolactone for the delivery of mitomycin C to bladder tu- Sci. 2016;11:404. mors. Int J Pharm. 2009;371:170. 32. Nanda AK, Wicks DA. The influence of the ionic concentration, concentration of the polymer, degree of neutralization and chain 13. Payyappilly SS, Panja S, Mandal P, Dhara S, Chattopadhyay S. extension on aqueous polyurethane dispersions prepared by the Organic solvent-free low temperature method of preparation for acetone process. Polymer. 2006;47:1805. self-assembled amphiphilic poly(ɛ-caprolactone)–poly(ethylene 33. Goloub T, Pugh RJ. The role of the surfactant head group in the glycol)block copolymer based nanocarriers for protein delivery. emulsification process: single surfactant systems. J Colloid Colloids Surf B: Biointerfaces. 2015;135:510. ɛ Interface Sci. 2003;257:337. 14. Sinha VR, Bansal K, Kaushik R, Kumria R, Trehan A. Poly- - 34. Hasani F, Pezeshki A, Hamishehkar H. Effect of surfactant and oil caprolactone microspheres and nanospheres: an overview. Int J type on size droplets of betacarotene-bearing nanoemulsions. Int J Pharm. 2004;278:1. Curr Microbiol App Sci. 2015;4:146. 15. JameelaSR,SumaN,MisraA,RaghuvanshiR,GangaS, 35. Mu L, Feng SS. A novel controlled release formulation for the ɛ Jayakrishnan A. Poly ( -caprolactone) microspheres as a vaccine anticancer drug paclitaxel (Taxol®): PLGA nanoparticles contain- carrier. Curr Sci. 1996;70:669. ing vitamin E TPGS. J Control Release. 2003;86:33. 16. Youan B, Benoit MA, Rollmann B, Riveau G, Gillard J. Protein-loaded 36. Feng S, Huang G. Effects of emulsifiers on the controlled release of poly (ɛ-caprolactone) microparticles. II. Muramyl dipeptide for oral paclitaxel (Taxol®) from nanospheres of biodegradable polymers. J controlled release of adjuvant. J Microencapsul. 1999b;16:601. Control Release. 2001;71:53. 17. Bouchemal K, Briançon S, Perrier E, Fessi H. Nano-emulsion for- 37. Jódar-Reyes AB, Martín-Rodríguez A, Ortega-Vinuesa JL. Effect mulation using spontaneous emulsification: solvent, oil and surfac- of the ionic surfactant concentration on the stabilization/ tant optimisation. Int J Pharm. 2004;280:241. destabilization of polystyrene colloidal particles. J Colloid 18. Lopez-Montilla JC, Herrera-Morales PE, Pandey S, Shah DO. Interface Sci. 2006;298:248. Spontaneous emulsification: mechanisms, physicochemical as- 38. Furusawa K, Sato A, Shirai J, Nashima T. Depletion flocculation of pects, modeling, and applications. J Dispers Sci Technol. latex dispersion in ionic micellar systems. J Colloid Interface Sci. 2002;23(1–3):219. 2002;253:273.